Poster abstracts
Poster number 44 submitted by Debadrita Paul
Investigating the unexpected connection between promoter proximal RNA Pol II pausing and nonsense-mediated mRNA decay
Debadrita Paul (Molecular Cellular and Developmental Biology MCDB), Rene Arvola (Depratment of Molecular Genetics), Guramrit Singh (Department of Molecular Genetics)
Abstract:
Nonsense-mediated mRNA decay (NMD) pathway degrades aberrant transcripts containing premature termination codons (PTCs) and regulates ~10% of the human transcriptome. A key NMD activator is the exon junction complex (EJC) that is co-transcriptionally deposited upstream of exon junctions during pre-mRNA splicing. The core NMD machinery, including UPF3 engages with the EJC present downstream of a PTC to trigger NMD. Unexpectedly, we discovered that the human UPF3 proteome includes components of the Super Elongation Complex (SEC), which phosphorylates RNA Pol II to release it from promoter-proximal pausing to enable transcription elongation. Further, depletion of MLLT1, an SEC component, significantly downregulates PTC-containing mRNAs. Conversely, knockdown of ICE1, a component of the Little Elongation Complex (LEC) that competes with SEC, upregulates NMD targets. To assess the role of SEC and RNA Pol II pausing in NMD, we have optimized a 4-thio-uridine pulse labelling based assay to compare the decay rates of specific PTC-containing mRNAs with their PTC-less counterparts from the same gene. Promoter-proximal pausing enhances binding of the nuclear cap-binding complex (CBC) to nascent pre-mRNAs. A recent study found that pre-EJC engages with transcription start site (TSS) to prevent RNA Pol II phosphorylation and its transition into elongation, but how pre-EJC is deposited remains unknown. We propose that the transcription and export (TREX) complex, which binds near mRNA 5’-ends and engages with both CBC and EJC, facilitates pre-EJC deposition near the TSS independently of splicing. Such pre-EJC deposition may cooperatively promote downstream EJC occupancy, thereby enhancing NMD. To test this proposal, we will disrupt CBC-TREX and TREX-EJC interactions and assess global EJC deposition, Pol II phosphorylation status and promoter-proximal pausing. Overall, we aim to reveal the mechanisms linking transcriptional elongation in the nucleus to mRNA decay in the cytoplasm.
References:
Akhtar, J., Kreim, N., Marini, F. et al. Promoter-proximal pausing mediated by the exon junction complex regulates splicing. Nat Commun 10, 521 (2019). https://doi.org/10.1038/s41467-019-08381-0
Watson M, Park Y, Thoreen C. Roadblock-qPCR: A simple and inexpensive strategy for targeted measurements of mRNA stability. RNA. 2020 Dec 7;27(3):335–42. doi: 10.1261/rna.076885.120. Epub ahead of print. PMID: 33288682; PMCID: PMC7901842.
Keywords: nonsense mediated mRNA decay, promoter proximal pausing, EJC